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DEVELOPMENTAL DYNAMICS 226:257–267, 2003
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A PEER REVIEWED FORUM
Cnidarians: An Evolutionarily Conserved Model
System for Regeneration?
T.W. Holstein,1* E. Hobmayer,1,2 and U. Technau1
Cnidarians are among the simplest metazoan animals and are well known for their remarkable regeneration capacity.
They can regenerate any amputated head or foot, and when dissociated into single cells, even intact animals will
regenerate from reaggregates. This extensive regeneration capacity is mediated by epithelial stem cells, and it is
based on the restoration of a signaling center, i.e., an organizer. Organizers secrete growth factors that act as
long-range regulators in axis formation and cell differentiation. In Hydra, Wnt and TGF-beta/Bmp signaling pathways
are transcriptionally up-regulated early during head regeneration and also define the Hydra head organizer created
by de novo pattern formation in aggregates. The signaling molecules identified in Cnidarian regeneration also act in
early embryogenesis of higher animals. We suppose that they represent a core network of molecular interactions,
which could explain at least some of the mechanisms underlying regeneration in vertebrates. Developmental
Dynamics 226:257–267, 2003. © 2003 Wiley-Liss, Inc.
Key words: Cnidaria; Hydra; regeneration; organizer; self organisation; wnt/wg signaling
Received 17 September 2002; Accepted 31 October 2002
INTRODUCTION
The freshwater polyp Hydra, a member of the ancient phylum Cnidaria,
is famous for its regenerative capacity. Just like the multiheaded monster
in Greek mythology that grew two
new heads for every one cut off, a
cnidarian polyp can regenerate a
new head after decapitation. Cnidarians are among the simplest living metazoans and evolved approximately 700 million years ago (Bridge
et al., 1995; Conway Morris, 2000;
Nielsen, 2001; Petersen and Eernisse,
2001). They consist of two body layers, an outer ectoderm and an inner
endoderm, separated by an extracellular matrix (mesoglea), and they
represent the first animals with a defined body axis and a nervous system.
1
The regenerative capacity of cnidarians is remarkable. Hydra polyps
can be dissociated into single cells
that can regenerate as reaggregates into an intact animal within a
few days. Cnidarian regeneration
occurs by morphallaxis, i.e., a process of repatterning of the existing
tissue without the necessity of cell
proliferation. This appears to be fundamentally different from regeneration in vertebrates, where wound
closure is followed by blastema formation during which cells beneath
the wound epidermis dedifferentiate, start to divide, and transdifferentiate (Lo et al., 1993; Brockes,
1997). However, recent data indicate that regeneration of cnidarian
tissue shares more similarities to vertebrate (urodele) regeneration than
previously thought. In this review, we
focus on the molecular regulation of
Hydra head regeneration in comparison to vertebrate systems. A
comprehensive treatment of classic
transplantation experiments, theoretical models, and molecular data
in Hydra is found in the review of H.
Bode (this issue).
At present, it is unclear to what
extent “adult” stem cells are involved in the regeneration process.
Such stem cells have been found
even in some mammalian tissues,
and they have a capacity for developing into a limited number of different cell types (for review, see Stocum, 2001). Of interest, stem cells in
cnidarians also mediate the morphogenetic plasticity of the tissue.
There are two epithelial stem cell
Department of Biology, Darmstadt University of Technology, Darmstadt, Germany
Zoological Institute, University of Innsbruck, Innsbruck, Austria
Grant sponsor: Deutsche Forschungsgemeinschaft.
*Correspondence to: Thomas W. Holstein, Molekulare Zellbiologie, Technische Universität, Schnittspahnstr. 10, 64287 Darmstadt,
Germany. E-mail: [email protected]
2
DOI 10.1002/dvdy.10227
© 2003 Wiley-Liss, Inc.
258 HOLSTEIN ET AL.
populations, an ectodermal and an
endodermal one, which continuously differentiate into head- and
foot-specific
tissue,
which
is
sloughed off at both ends of the
body axis (Campbell, 1967a,b;
David and Campbell, 1972). A third
stem cell system that is present at
least in Hydra and some other Hydrozoans gives rise mainly to nerve
cells, nematocytes, and gland cells,
but it is not required for regeneration
(David and Gierer, 1974).
In all regenerating tissue, a major
question is what instructs the cells involved in regenerative processes
and which gene products are responsible for the induction of regeneration. The application of molecular and genetic techniques has
shown that several crucial genes of
early embryogenesis is evolutionarily
conserved between vertebrates
and insects. Although little is known
to date about cnidarian embryogenesis on the molecular level, new
molecular data indicate that some
of the homologous genes involved
in bilaterian embryogenesis act during cnidarian regeneration. Therefore, the intriguing possibility exists
that a common set of genes might
control at least the early steps of the
regeneration process in cnidarians
and bilaterians. We presume that
the high or even unlimited regenerative capacity characteristic for
cnidarians reflects the properties of
an ancient patterning system that
can generate complete structures
(whole organisms), starting from a
broad range of initial conditions. It is
plausible that molecular patterning
systems capable of extremely robust
and flexible self-organisation might
have been selected during early
metazoan evolution and became
conserved in higher animals. This review, therefore, mainly emphasizes
the cellular and molecular dynamics
of this self-organisation system during regeneration, and it represents
essentially one lab’s view of the
problem. We presume that the signaling molecules identified in cnidarian regeneration represent a
core network of molecular interactions that could be responsible for at
least some of the mechanisms underlying regeneration in vertebrates,
e.g., limb regeneration (Brockes,
1997; Gardiner et al., 1999).
CNIDARIAN’S REGENERATIVE
CAPACITY IS BASED ON A
HIGH MORPHOGENETIC
PLASTICITY OF THE TISSUE
Epithelial Stem Cells
Most cnidarian polyps and even
some medusae propagate asexually, so that they are in a steady
state of constant growth and tissue
turnover. In Hydra polyps, it has
been shown that both layers of the
body wall, the ectoderm and the
endoderm, are comprised by dividing epithelial stem cells in which
newborn cells are passively displaced upward to form the stinging
tentacles, downward to form the
foot, or bud off at the sides to make
replica animals (Campbell, 1967a,b;
for review see Bode and Bode,
1984). An important consequence is
that the passively displaced cells
have to assess their relative position
in the organism continuously.
Hence, patterning systems necessary to provide this information are
continuously active in Hydra polyps.
By contrast, in mammals, most of
these morphogenetic signals are
mainly active only during embroygenesis.
During regeneration, these morphogenetic signals can be activated or enhanced at the site of
wounding. Figure 1 summarizes the
major events during the regeneration process in Hydra and other Cnidarians. When Hydra is cut in half,
the upper half containing the head
will regenerate a new foot, and the
lower half containing the foot will regenerate a new head. Regeneration is a rapid process. After wound
closure, which takes approximately
1–3 hr, the tentacles of a new head
differentiate within 36 hr and a regenerating foot becomes sticky
again within 30 hr (Hoffmeister and
Schaller, 1985). Far less is known
about foot regeneration, yet the
mechanisms of head and foot regeneration are probably similar, although there is some evidence that
the head system has some supportive function for the foot system (Müller, 1990, 1995, 1996; Lee and Javois,
1993; Forman and Javois, 1999;
Javois and Frazier-Edwards, 1991;
Schiliro et al., 1999). However, the
molecular basis for this phenomenon is completely unclear. If a Hydra
is cut into several pieces, the middle
portions will regenerate both heads
and feet at their appropriate ends,
maintaining the initial polarity (Marcum et al., 1977). By comparison, an
isolated foot or head alone cannot
regenerate an intact animal, only if
a head is transplanted on a foot, the
missing body region will be intercalated (Holstein and David, 1990).
Morphallaxis or Epimorphosis?
Classic experiments using Hydra polyps that were either x-ray irradiated
(Hicklin and Wolpert, 1973; Noda
and Egami, 1975) or treated with the
S-phase blocking agent hydroxyurea before regeneration have
shown that cell division is not required for the formation of a new
head (Cummings and Bode, 1984).
This finding has led to the conclusion
that regeneration in Hydra is morphallactic (Gilbert, 2000; Wolpert,
2002). However, the cellular dynamics appear to be more complicated.
Figure 2A shows that, 12 hr after
head removal, the regenerating tip
is completely free of S-phase cells
(Park et al., 1970; Holstein et al., 1991;
Fig. 2B), but by 30 hr, the pattern has
completely changed, and the regenerating tip is more strongly labeled than the gastric tissue (Fig.
2C). This finding demonstrates dramatic effects on the cell cycle and
proliferation at the regenerating site.
Whether the resumption of mitosis is
due to a decay of the inhibitory effect or a release of a stimulatory signal is not clear. Molecules of the extracellular matrix (ECM) also appear
to contribute to the changes in the
proliferation patterns during Hydra
head regeneration (Sarras et al.,
1991, 1993; Yan et al., 1995, 2000;
Shimizu et al., 2002). Accordingly, a
loss of the ECM could be related to
the reduced mitotic activity and a
restoration of the ECM to the delivery of mitogenic factors. Of interest,
newborn buds also exhibit a dramatic increase in cell proliferation,
and the head region is characterised by a continuous high level of
CNIDARIAN REGENERATION 259
Fig. 2. Changes in the pattern of epithelial
cell cycling during head regeneration. A:
Interstitial cell-free polyps were cut at 60 –
70% body length, pulse-labeled with bromodeoxyuridine (BrdU) at the times indicated, and processed for BrdU visualization
by indirect immunofluorescence (camera
lucida drawings). B,C: Photomicrographs illustrating the pattern of epithelial cell proliferation during head regeneration at 12 hr
(b) and at 36 hr (c). 姝 Academic Press (from
Holstein et al., 1991).
Fig. 1. Major processes during cnidarian regeneration on the cellular and molecular
level. The minimal steps necessary for cnidarian regeneration (solid arrows) involve (1)
wound closure of the epithelia followed by (2) the establishment of an organizer and the
instruction of epithelial stem cells, and (3) subsequent morphogenesis. There are also
nonobligatory steps in cnidarian regeneration (dotted arrows), e.g., dedifferentiation in
terminally differentiated tissue of medusae (Schmid, 1992) and cell proliferation in Hydra
polyps (Holstein et al., 1991), which could be related to the blastema formation in
vertebrates.
proliferating cells (Holstein et al.,
1991). This finding indicates that the
loss and restoration of ECM upon
wounding is not the only prerequisite
for the effect on the cell proliferation
during regeneration. It is worth noting that a similar correlation between regeneration and proliferation was also found in other
cnidarians, e.g., the Cubopolyp
Carybdea marsupialis (Holstein and
Stangl, manuscript in preparation).
Factors that stimulate mitosis of
epithelial cells have been described. Schaller and coworkers
(Schaller, 1976; Schaller et al., 1977,
1990) have shown that, if Hydra is
treated with low concentrations of a
neuropeptide, the head activator,
mitosis is stimulated in epithelial cells.
A local release of this factor could
lead to an increased level of mitosis
during regeneration. Hobmayer et
al. (1997) demonstrated that head
activator treatment stimulates epithelial cell division and induces the
formation of more tentacle-specific
epithelial cells during regeneration.
Consistent with these findings, we
also found that inhibition of cell
proliferation by aphidicolin or hydroxyurea treatment leads to an in-
Fig. 3. Local up-regulation of Hy␤-Cat,
HyTcf, and HyWnt expression in head regenerating tips. Whole-mount in situ hybridizations at 1 and 48 hr after head removal.
The Wnt signaling cascade is up-regulated
during head regeneration. HyWnt is expressed in a group of 15–50 cells defining
the head organizer region at the tip of the
hypostome. 姝 MacMillan Press (from Hobmayer et al., 2000).
260 HOLSTEIN ET AL.
complete regeneration of head
structures after 36 hr (Hobmayer and
Holstein, unpublished observations).
Therefore, we presume that morphogenetic signals and growth factors
that are released during head regeneration in cnidarians can also induce cell proliferation, which is required for a complete regeneration
of the full-sized structure (Fig. 1).
Dedifferentiation as a
Necessary Step in
Regeneration?
Although most polyps grow constantly, medusae normally exhibit
only a limited capacity to grow,
which is related to the differentiation
of a sexually mature animal (there is
an exception from this rule, because
in some hydrozoan life cycles, medusae can propagate asexually,
e.g., Rathkea and Sarsia; for review,
see Tardent, 1978). Nevertheless,
even terminally differentiated medusa tissue can regenerate (Fig. 1).
Pioneering studies of the jellyfish
Podocoryne carnea showed that
the muscle tissue of adult medusae
transdifferentiates into several different cell types during regeneration
(for review, see Schmid, 1992;
Schmid and Reber-Müller, 1995).
When striated muscle cells were explanted from the subumbrella and
cultured in the presence of ECM degrading enzymes, diacylglycerol, or
the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, they dedifferentiated and started to proliferate 24 – 48 hr later (Schmid et al.,
1998). Such cells formed flagellae
first, which can be interpreted as a
sign for the naive state of this tissue,
and later they differentiated into
smooth muscle cells, sensory cells, or
nematocytes (Alder and Schmid,
1987). During transdifferentiation
several mesodermal genes, which
are specific for striated muscle differentiation, were turned off (Müller et
al., 1999; Yanze et al., 1999; Spring et
al., 2000). This finding is in accord
with the morphologic data.
Cnidarian vs. Vertebrate
(Urodele) Regeneration
A comparison of the basic features
of cnidarian regeneration with the
regeneration process in vertebrates
indicates that both systems share
some common principles, despite
the prevailing view that one is morphallactic (Cnidaria) and one is epimorphic (vertebrates). The remarkable regeneration capacity of
urodele amphibians involves the local dedifferentiation of stump tissue
to form a blastema and new growth
within the blastema to form distal
structures (for review, see Gardiner
and Bryant, 1996; Brockes, 1997;
Gardiner et al., 1999; Wolpert, 2002).
Yet, recent elegant molecular and
genetic data suggests that limb bud
formation and regeneration in vertebrates involves a prepatterning of
the whole limb at an early stage in a
small morphogenetic field (prespecification model), rather than a
distal transformation (progress zone
model) of the growing blastema
(Sun et al., 2002; Dudley et al., 2002;
Duboule, 2002). This finding suggests
that, also in vertebrate limb formation and regeneration, patterning of
a morphogenetic field occurs only
at small scale and growth is needed
only to add in cells to produce a
structure of larger size. The reason for
this may lie in the fact that morphogens can act only over a distance of
several cell diameters and a maximum of 300 ␮m.
In cnidaria, neither local dedifferentiation nor blastema formation
are obligatory steps in the regeneration process, which is probably due
to the high morphogenetic plasticity
of the tissue. Epithelial stem cells are
always competent for morphogenetic signals released at the site of
wounding, and they can differentiate even when cell cycling is
blocked. Hence, it appears that, in
Hydra, cell division is not completely
indispensable for the regeneration
of the complete structure. On the
other hand, in vertebrates, growth of
the blastema might only be necessary to enlarge an already patterned field. Thus, Hydra and vertebrate regeneration might share
more features than commonly
thought. The crucial and obligatory
step during regeneration in both systems is a prepatterning of the regenerating tissue. Regeneration always
gives rise to structures with positional
values proximal to the site of regen-
eration. In cnidarians there is substantial evidence that an apical signaling center, the head organizer, is
the driving force for this process. In a
first step, this organizer has to be reestablished at the regenerating tip.
Then, signals emanating from this organizing center pattern and respecify the tissue proximal to the
wounding site. This emphasizes the
importance of the reestablishment
of an organizer during the initial
steps in regeneration. In the following, we will discuss the molecular
features of the Hydra head organizer and how it is reestablished during regeneration, particularly in reaggregates.
HEAD REGENERATION IN HYDRA
IS DRIVEN BY THE RESTORATION
OF AN APICAL SIGNALING
CENTER, THE HEAD ORGANIZER
The capacity to regenerate a head is
higher at the apical end than at the
basal end of Hydra’s body axis (Webster, 1966a,b; Wilby and Webster,
1970a,b; Wolpert et al., 1971, 1972;
MacWilliams, 1983b; Technau and
Holstein, 1995). Transplantation experiments have shown that the peak of
this activity is localized in the hypostome. A small piece of tissue from the
hypostome induces a secondary
body axis when grafted laterally to
another polyp (Browne, 1909; Mutz,
1930; Yao, 1945; Broun and Bode,
2002). Hence, in terms of organizer activity, Hydra’s hypostomal tissue is
equivalent to the dorsal lip of the frog
embryo, the Spemann-Mangold organizer, which also induces a secondary body axis when grafted to the
ventral side of the embryo (Spemann
and Mangold, 1924).
Until recently, it was rather unclear
when and how the organizer and its
molecular composition arose during
animal evolution (Harland and Gerhart, 1997; Knoll and Caroll, 1999).
However, the discovery that the
same set of genes is active in the
organizer of all vertebrates suggested that basic features of signaling centers acting as an organizer
might have arisen earlier in metazoan evolution. Potential signaling
molecules that could act as diffusible morphogens similar to those in
vertebrates have been identified re-
CNIDARIAN REGENERATION 261
Fig. 4. Regeneration of intact Hydra polyps from reaggregates of dissociated single cells. Formation of the tissue bilayer by
ectoderm- and endoderm-specific cell sorting mechanisms is finished within 24 hr. Development of polyp structures occurs within
96 hr.
cently in Hydra (Hobmayer et al.,
2000). It is likely that the gradient of
inductive capacity is mediated by a
gradient of these signaling molecules released from the Hydra head
organizer.
A Wnt ligand (HyWnt) and the cytoplasmic mediators Dishevelled (HyDsh),
GSK3 (HyGSK3), and ␤-Catenin (Hy␤
-Cat) were cloned from Hydra (Hobmayer et al., 2000). In a two-hybrid
screen with Hydra ␤-Catenin as bait,
the transcriptional coactivator Tcf
(HyTcf) was also identified (Hobmayer et al., 2000). A Hydra member
of the family of Frizzled receptors
was identified by Minobe et al.
(2000). Hence, the core Wnt pathway is present in Hydra. In situ hybridization revealed that Wnt signaling
acts in axial patterning and during
head regeneration in Hydra. HyWnt
is expressed in a small number of ectodermal and endodermal epithelial
cells in the apical tip of the hypostome, which represents the Hydra
head organizer. HyTcf expression is
also restricted to the hypostome of
the polyp, but the HyTcf expression
domain is broader than the HyWnt
spot encompassing the entire hypostome and, thereby, possibly demarcating the range of action of
the HyWnt ligand (Hobmayer et al.,
2000). During head regeneration,
HyWnt, HyTcf, and Hy␤-cat are
among the earliest genes to be upregulated, within 30 – 60 min after
wound healing (Fig. 3). In the budding zone, where the new body axis
of the daughter polyp is initiated
(Otto and Campbell, 1977), activation of the HyWnt pathway also starts
Fig. 6. Expression dynamics of HyTcf, HyWnt, and HyBra1 during aggregate development. In situ hybridization reveals patterning events during head organizer
formation. HyWnt and HyBra1 appear simultaneously in small spots (24 hr). which enlarge during later stages (96 hr), and precede formation of morphologic head
structures by approximately 2–3 days. All
spots eventually develop into heads. 姝 Proceedings of the National Academy of Sciences USA 2000;97:12127–12131 and Nature
2000;407:186 –189.
Fig. 5. Head induction by activated cell
clusters. A: A two-headed aggregate (96
hr) with a head containing a green-labeled
60-␮m cluster of aggregated cells from dissociated 12-hr regenerating tip tissue. B: Efficiency of cell clusters to induce head formation. Head formation frequency of
single cells (30 ␮m) and different cell cluster
sizes were scored 80 hr after aggregation in
carrier tissue derived from whole polyps
(filled circles, hatched line) and polyps
lacking the upper fifth (filled triangles, solid
line; P ⬍ 0.001; n ⫽ 28 –53; means [SEM,
three experiments]). Control clusters derived from the corresponding carrier tissue
are indicated by open circles and open
triangles. C: Effect of head inhibition in aggregates. Aggregates that contained competing 120-␮m and 60-␮m cell clusters in a
single aggregate. A 120-␮m cell cluster inhibited the formation of a head, the 60-␮m
cell cluster; the correlation of head formation frequency of 60 ␮m cell clusters with
their distance from the nearest head is
shown. 姝 A–C from Proceedings of the National Academy of Sciences USA 2000;97:
12127–12131. Scale bar ⫽ 200 ␮m in A.
with an up-regulation of Hy␤-Cat
and HyTcf and is followed by HyWnt
expression in a spot of 10 –15 cells
(Hobmayer et al., 2000). These data
indicate a pivotal role for the members of the Wnt-pathway in setting
up the Hydra head organizer.
There is also evidence for a second major signaling system in cnidarians, i.e., the TGF␤/Bmp signaling
pathway and its antagonist Chordin
(Samuel et al., 2001; Lelong et al.,
2001; Hobmayer et al., 2001; Hayward et al., 2002), which are involved in early embryonic axis formation of vertebrates. A Bmp ligand
(Reinhardt and Bode, personal communication), a highly conserved receptor-regulated Smad1 homologue (Hobmayer et al., 2001a), and
the Bmp antagonist Chordin (Rentzsch, Hobmayer, and Holstein, unpublished observations) have been
found in Hydra. The expression patterns of HySmad1 and Chordin during regeneration are consistent with
the hypothesis that Bmp signaling is
suppressed by Chordin, which would
indicate a conservation of the molecular interactions of dorsoventral
patterning from Hydra to vertebrates (for review, see DeRobertis
and Bouwmeester, 2001; Shilo, 2001).
These data demonstrate that at
least two major signaling systems
that are responsible for the function
262 HOLSTEIN ET AL.
of the vertebrate organizer (DeRobertis and Sasai, 1996; DeRobertis and
Bouwmeester, 2001) are already
present in Hydra. This finding suggests that the core Wnt signaling
pathway as well as the TGF␤/Bmp
signaling pathway and its antagonist Chordin were present in the
common ancestor of diploblastic
cnidarians and the triploblastic Bilateria and, hence, most likely were a
basic feature of early multicellular
animals. Notably, a TGF-beta receptor was found in sponges (Suga et
al., 1999), although its expression is
unclear to date.
It should be also pointed out that
transcription factors that play a role
in the vertebrate organizer have
been isolated from Hydra, such as
the HNF3␤ homolog budhead (Martinez et al., 1997), the homeobox
gene goosecoid (Broun et al., 1999),
and the T-box gene Brachyury
(Technau and Bode, 1999). These
genes are all expressed in the organizer region in Hydra (for review, see
Galliot, 2000) and may have a function in regulatory feedback loops
together with the Wnt and TGF␤ signaling cascades during head regeneration.
REGENERATION OF THE HEAD
ORGANIZER FROM
REAGGREGATED SINGLE CELLS
Hydra can be completely dissociated into single cells and will regenerate intact animals within 3 to 4
days (Fig. 4) (Noda, 1971; Gierer et
al., 1972). After dissociation into a
single cell suspension and subsequent reaggregation, all existing
gradients of the polyp and any positional information are destroyed
and have to be reestablished
(Gierer et al., 1972; Sato et al., 1992;
Technau and Holstein, 1992). This experimental system is unique in that it
is possible to analyze regeneration
from the very beginning and under
conditions of de novo pattern formation on the cellular and molecular level.
Reaggregation proceeds through
a well-defined sequence of morphogenetic processes: initial cell adhesion, ecto– endo cell sorting, formation of the epithelial bilayer,
differentiation of a head and foot,
and finally separation into intact
polyps. To establish the epithelial bilayer configuration, three interaction
types are necessary: ecto– ecto,
endo– endo, and ecto– endo cell interactions. The formation of homotypic ectodermal and homotypic
endodermal aggregates was first
observed during rotary culture of dissociated cell suspensions (Technau
and Holstein, 1992) and confirmed
by laser-cell trapping experiments,
where the adhesive forces between
individual cells were directly determined (Sato-Maeda et al., 1994).
Pairs of endodermal cells exhibited
stronger adhesive forces than pairs
of ectodermal epithelial cells, and
there was no initial heterotypic interaction between individual ectodermal and endodermal cells. Hobmayer et al. (2001b) used rotary
culture of dissociated cell suspensions and found that aggregation of
epithelial cells proceeded in two
steps: first homotypic (ecto– ecto
and endo– endo) interactions created small cell clusters, then heterotypic interactions between ectodermal and endodermal cell clusters
led to the formation of larger aggregates. This switch from homotypic to
heterotypic interaction occurred at
a critical aggregate size of 10 –20
epithelial cells and indicates that
adhesive forces between ectodermal and endodermal cells became
significantly stronger than adhesive
forces between either ectodermal
or endodermal cells (Hobmayer et
al., 2001b). At present it is unclear
whether this change in the cell– cell
affinities in Hydra reaggregates can
be explained by a depletion of a
limited pool of cell adhesion molecules, a redistribution and clustering
of preexisting heterotypic adhesion
molecules (Grawe et al., 1996), or
the new expression of heterotypic
adhesion molecules due to an activation of intracellular signaling cascades in homotypic aggregates
(Fagotto and Gumbiner, 1996).
The formation of ecto– endodermal cell clusters finally leads to the
formation of ectodermal and
endodermal tissue layers. During this
epithelial sheet formation, the ectodermal tissue layer begins an epiboly-like movement to spread over
the endoderm (Kishimoto et al.,
1996). In parallel, the endodermal
layer organizes beneath an intact
ectodermal layer (Murate et al.,
1997), suggesting that the formation
of both epithelial layers is driven by
the ectoderm. This dramatic process
of cell sorting and restoration of cell
polarity are completed within the
first 12 hr of the reaggregation process (Gierer et al., 1972; Technau
and Holstein, 1992). Once the ectodermal and endodermal layers are
established, no further rearrangement occurs. With respect to their
original axial position in Hydra, no
cell sorting has been observed (Sato
et al., 1992; Technau and Holstein,
1992). This finding indicates that, after dissociation into single cells, there
is no predisposition of erstwhile head
cells to sort out into head tissue and
that the formation of new activation
centers and head organizers occurs
by true de novo pattern formation
(Gierer et al., 1972; Technau and
Holstein, 1992).
In further experiments, it was shown
that a community effect regulates the
formation of activation centers in Hydra (Technau et al., 2000). Labeled
cell clusters were produced from regenerating stumps that have a high
competence for head induction
(MacWilliams, 1983b). The regenerating tissue was dissociated into single
cells, aggregated in rotary culture,
and the resulting cell clusters were
fractionated by size (Technau et al.,
2000). Small labeled cell clusters consisting of 10 –15 cells (60 ␮m in diameter) were added to an unlabeled cell
suspension, and approximately half of
them were found to be present in a
developing head after reaggregation. The labeled cells were confined
to the hypostome while the tentacles
were formed by the host tissue (Fig.
5A,B). This finding shows that a cluster
of only 10 to 15 cells is necessary and
sufficient to instruct and recruit surrounding host tissue and initiate the
formation of a new head, which is the
definition of an organizer sensu strictu.
Single cells or very small clusters (30
␮m in diameter) consisting of a few
epithelial cells have virtually no elevated capacity of induction. These
data demonstrate that a community
effect (Gurdon et al., 1993) between
these cells is essential to create a stable signaling center. Further experi-
CNIDARIAN REGENERATION 263
Fig. 7. Model of putative positive feedback in Hydra Wnt signaling. Preliminary
evidence and comparison with higher
metazoans support the view that autocatalytic self-activation of the Wnt-pathway and
a feedback between HyWnt and the transcription factor HyBra1 are involved in establishment and maintenance of the HyWnt
signaling cascade (multiple arrows).
ments indicated an activation range
of approximately 45 ␮m (two to three
epithelial cell diameters; Technau et
al., 2000), which is in the estimated
diffusion range of known morphogens, i.e., wnt/wingless in Drosophila
(Gurdon and Bourillot, 2001; see below).
AUTOCATALYTIC SHORT-RANGE
HEAD ACTIVATION DURING
REGENERATION BY THE WNTPATHWAY?
That small clusters of cells can induce surrounding tissue to differentiate into head tissue suggests that
diffusible morphogens like Wnt might
play an instructive role in the activation process. The expression pattern
of HyWnt was examined in early reaggregates (Hobmayer et al., 2000;
Technau et al., 2000) and found to
occur in small spots comprising only
a few epithelial cells (Fig. 6) by 24 hr.
At this time, cells have completely
sorted out into ectodermal and
endodermal layers (Gierer et al.,
1972; Technau and Holstein, 1992),
indicating that HyWnt activation requires intact epithelial tissue. By
96 hr, the HyWnt expression domains
have enlarged to their final size in
future hypostomes (Fig. 6). The size of
early HyWnt spots is 50 – 60 ␮m,
which corresponds to the minimal
cluster size that can act as an organizer (see Fig. 5B).
Reaction-diffusion models of pattern formation predict an autocatalytic feedback loop during the activation process. Preliminary data
suggest a possible feedback control
in the HyWnt pathway (Fig. 7). Hy␤cat and HyTcf are expressed uniformly throughout aggregates and
later become restricted to domains
where new heads are being formed
(Fig. 6). A uniform, but high level of
HyTcf and Hy␤-Cat might provide a
competence to cells to produce HyWnt. Activation of HyWnt might be a
stochastic process which is initiated
in single cells, but only maintained if,
by chance, neighboring cells also
express HyWnt. Alternatively, HyWnt
might activate and stabilize its own
expression directly by means of its
transcriptional mediators Hy␤-Cat
and HyTcf and later become restricted to domains where new
heads are being formed. Notably,
the expression of HyWnt always preceded the apparent restriction of
domains in the initially symmetrical
environment of an aggregate, and
all HyWnt domains finally form a
head (Technau et al., 2000). Both
scenarios are consistent with the
idea of an autocatalytic feedback
loop and that HyWnt is a direct target gene of an active Hy␤-Cat/
HyTcf complex. This finding is in line
with findings from Drosophila, where
autocatalytic self-activation of Wg
and a functional Tcf-binding site in
the Wg promoter have been demonstrated (van de Wetering et al.,
1997; Lessing and Nusse, 1998).
There is additional evidence that
HyWnt might be coupled also by a
positive feedback with another early
head gene, HyBra1 (Fig. 7), a Hydra
homologue of the T-box gene
Brachyury (Technau and Bode,
1999). In aggregates, size and time
of appearance of small HyBra1positive spots are equivalent to the
HyWnt expression dynamics. Interestingly, HyBra1 also shows synexpression with HyWnt during budding
and head regeneration as well as in
adult polyps, although the HyBra1positive domain in the steady state
hypostome is broader than the HyWnt-positive domain (Technau and
Bode, 1999). A putative Tcf-binding
site has been identified recently in
the HyBra1 promoter (Technau, unpublished data), which supports the
idea that Brachyury and Wnt are
members of a synexpression group
in Hydra. In mouse embryos and
mouse cell lines, Brachyury is a direct
target gene of Wnt3a signaling (Liu
et al., 1999; Galceran et al., 2001),
and Brachyury itself activates transcription of Wnt11 in Xenopus (Tada
and Smith, 2000). Direct experimental proof for such a feedback loop in
Hydra by testing the effect of exogenous HyWnt on HyBra1 expression
or by loss-of-function experiments
with the HyBra1 gene would be of
particular importance.
SIZE CONTROL DURING
REGENERATION BY LONGRANGE INHIBITION
Patterning processes have to be restricted to the regenerating tissue.
On the theoretical level, reaction–
diffusion mechanisms (Turing, 1952)
predict that an inhibitor is produced
by the activation center and transmitted to the surrounding tissue to
prevent the initiation of another activation center (Gierer and Meinhardt, 1972; Meinhardt, 1982, 1993).
Transplantation experiments using
intact Hydra have provided strong
evidence for such an inhibitory gradient extending from head into body
column (MacWilliams, 1983a,b). The
range of inhibition in regenerating
aggregates was determined by introducing cell clusters of different
size into a host aggregate where
larger cell clusters (120 ␮m) exerted
an inhibitory influence on the smaller
clusters (60 ␮m). It was found that
approximately 50% of the small clusters were not involved in head formation at a distance of 600 ␮m from
the large clusters, whereas essentially all of them were in heads at
1,000 ␮m from the large clusters, indicating an effective range of inhibition of approximately 800 –900 ␮m
(Technau et al., 2000). By comparison, the range of activation was approximately 45 ␮m, hence, 20⫻
shorter, which fits with theoretical
predictions (Gierer and Meinhardt,
1972; MacWilliams, 1982).
The molecular nature of the inhibit-
264 HOLSTEIN ET AL.
ing gradient is currently unclear. Using
antibodies to the gap junction proteins, Fraser et al. (1987) could perturb
the head inhibition gradient in grafting operations, suggesting that the inhibition gradient is mediated by cell–
cell communication by means of gap
junctions. However, the inhibition gradient might also involve long-range
morphogens regulating the properties
of epithelial cells. In the Drosophila
wing disc and the amphibian blastula
animal cap (Day and Lawrence,
2000; Lawrence, 2001), members of
the TGF␤/Bmp family act as longrange morphogens up to 300 ␮m
and concentration-dependent effects have been confirmed (Gurdon
and Bourillot, 2001). Changes in production of Dpp, the Drosophila Bmp
homolog, can substantially redesign
the Drosophila wing, indicating a
long-range action. However, recent
studies using GFP-Dpp constructs suggest a more complex mode of gradient formation, including endocytotic
trafficking and degradation (Entchev
et al., 2000). The antagonistic factor to
Dpp/BMP2-4 is Sog/Chordin, which
forms an opposing gradient. In Xenopus embryos the Chordin gradient
can have a range of at least 450 ␮m
when overexpressed, although its in
vivo range, which is restricted by the
metalloprotease Xolloid, appears to
be smaller (Blitz et al., 2000). Recently,
it has been shown directly in Drosophila that Sog forms a protein gradient in
dorsal cells of the embryo (Srinivasan
et al., 2002). On the dorsal side, Tolloid
(Tld) degradation and a dynamin-dependent retrieval of Sog act as a dorsal sink for active Sog (Srinivasan et al.,
2002). This long-range activity of Sog/
Chordin and the related degradation
by Tolloid/Xolloid could be an important component of the long-range inhibition phenomena and size control
of Hydra during regeneration.
PERSPECTIVE
Earlier work has suggested that regeneration by morphallaxis (as found in
Hydra) and epimorphosis (as found in
vertebrates) are fundamentally different. New experimental results from Hydra and vertebrates reveal, however,
that regeneration in these evolutionarily extremely distant phyla share
some similarities (see Fig. 1).
In an initial phase of regeneration,
after wound closure, epithelial stem
cells can respond to changes of
patterning signals at the wounding
site. If the cells are differentiated, as
is the case in medusae of Podocoryne (Schmid, 1992), they first have
to dedifferentiate to adopt a new
fate. However, this dedifferentiation
appears not to be an obligatory
step, as there is no evidence for it in
Hydra. Because all epithelial stem
cells along the body column of Hydra are competent to respond to
the regeneration signal, it is an important and unsolved question
whether this locally restricted response is due to a positive stimulatory signal or to a release of an inhibitory signal at the regenerating
site.
In the next phase of regeneration,
i.e., the formation of an organizer
and the establishment of a prepattern, substantial progress has been
made on the molecular level. It is
striking to note that a set of highly
conserved genes, i.e., the Wnt and
TGF-beta pathways as well as members of the T-box gene family, are
involved in cnidarian regeneration.
The data reviewed here indicate
that Hydra, a representative of one
of the oldest metazoan phyla, uses
these genes in a signaling center for
regulating the establishment and regeneration of its major body axis.
These genes also have a crucial role
in the patterning of higher animals.
This finding indicates the antiquity of
this patterning system and points toward an origin of signaling centers in
the earliest multicellular animals.
Eventually, patterning signals have
to be translated into morphogenesis
and differentiation of cells. (Non-canonical) Wnt-signaling and the T-box
transcription factor Brachyury are
good candidates for mediating patterning to morphogenesis. In chordates, Brachyury is a target gene of
Wnt, TGF-␤, and FGF signaling and a
transcriptional activator of many
genes involved in convergence and
extension, cell adhesion, and cytoskeleton (Tada et al., 1998; Takahashi et al., 1999; Tada and Smith,
2000).
At present, we are far away from a
comprehensive view of the genetic
network controlling regeneration
and the reestablishment of a body
axis in cnidarians. Genomic approaches, screens to identify the extracellular antagonists of signaling
molecules, and promotor analyses
of the involved genes will help to
understand this genetic network. This
progress will lead to the identification of cell-type–specific downstream genes, and to a better understanding of the question to what
extent cell proliferation is involved in
the cnidaria regeneration.
Another open question, not addressed in this review, is how peptide
signaling molecules are related to
the known signal transduction pathways. These peptides also affect cnidarian regeneration (Schaller, 1973;
Endl et al., 1999; Hampe et al., 1999;
see the review of Fujisawa in this issue) and may represent a phylogenetically ancient feature of cnidarians. However, it is totally unclear at
present to what extent these cnidarian peptides have homologues in
vertebrates. A gene encoding
members of the LWamide peptide
family, one of the peptide families
identified by the Hydra Peptide
Project (Takahashi et al., 1997; Bosch
and Fujisawa, 2001), has been identified recently in Caenorhabditis elegans (see review of Fujiswa in this
issue). Thus, signaling peptides easily
could have been overlooked with
algorithms that are normally used in
sequencing projects.
ACKNOWLEDGMENT
We thank C.N. David (Munich) for his
critical comments on the manuscript.
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